Anti-aging composition containing recombinant collagen and preparation method thereof

Abstract

The invention relates to an anti-aging composition containing recombinant collagen and a preparation method thereof. The main component of the composition is high-purity collagen obtained through a gene recombination technology, and various anti-aging active components such as sodium hyaluronate, vitamin C, coenzyme Q10 and vitamin E are combined. The invention also provides a preparation method, which ensures that each component is uniformly distributed and the biological activity is not damaged through low-temperature homogenization treatment and accurate proportioning. The composition disclosed by the invention can be used for remarkably promoting the proliferation of human skin fibroblasts and enhancing the expression of I-type collagen genes, and has a remarkable anti-aging effect. The composition is high in safety and remarkable in effect, is suitable for preventing and treating skin aging, and is widely applied to the fields of medicines and cosmetics.

Description

Technical Field

[0001] The invention relates to the fields of biological cosmetics and medicine, and in particular to an anti-aging composition containing recombinant collagen and a preparation method thereof. Background Art

[0002] Skin aging is a natural physiological process, but due to the accumulation of external environment (such as ultraviolet rays, pollution) and oxidative stress reactions in the body, the skin aging process is accelerated, manifested as wrinkles, sagging, spots and other aging characteristics. Collagen in the skin, especially type I collagen, is an important component for maintaining skin structure and elasticity, accounting for 70%-80% of the total protein in the dermis. With age, the body's ability to synthesize collagen gradually decreases, and at the same time, collagen fiber degradation intensifies, causing the skin structure to loosen and lose elasticity, and then forming wrinkles.

[0003] Currently, most of the collagen products used for anti-aging on the market are derived from animal tissues, such as cow, pig or fish skin extracts. Although these products have a certain effect in supplementing collagen, there are several problems: first, animal extracts have a potential risk of pathogen transmission, such as mad cow disease or viral infection; second, the structure of collagen from different species is different from human collagen, which can easily cause immune reactions and allergies; third, the purity of animal-extracted collagen is low, which affects its biological activity and effect.

[0004] In recent years, with the rapid development of genetic engineering technology, recombinant collagen has gradually attracted attention as a new type of skin anti-aging material. Humanized collagen expressed recombinantly in microbial expression systems (such as Escherichia coli, yeast or mammalian cells) can obtain collagen with higher purity and a structure closer to human collagen. In addition, the production process of recombinant collagen is stable and controllable, and is not affected by differences in animal sources and production batches, so it is safer and more consistent. More importantly, recombinant collagen has shown excellent efficacy in maintaining skin elasticity, promoting wound healing and reducing wrinkles.

[0005] However, single collagen supplementation often fails to achieve the best anti-aging effect. Skin aging is not only caused by collagen loss, but also accompanied by water loss, free radical damage and weakened skin barrier function. Therefore, the overall effect of anti-aging products can be enhanced by combining other ingredients that contribute to skin moisturizing, anti-oxidation and repair (such as hyaluronic acid, vitamin C and antioxidants). In view of this, the present invention proposes an anti-aging composition containing recombinant collagen, which aims to provide a safe and efficient collagen source through recombinant technology, and combines ingredients such as hyaluronic acid, vitamin C and antioxidants to further enhance its anti-aging effect. Summary of the invention

[0006] The invention relates to an anti-aging composition containing recombinant collagen and a preparation method thereof, belonging to the field of biomedicine and cosmetics, in particular to the development of a bioactive preparation for anti-aging, promoting collagen synthesis, improving skin elasticity and reducing wrinkles.

[0007] In recent years, as people pay more and more attention to skin care, anti-aging has become an important research direction in the field of beauty and skin care. Skin aging is mainly manifested in the loss of collagen, increased cellular oxidative stress and weakened skin barrier function. Traditional animal-derived collagen has limitations in application due to its complex source and easy to induce immune response. Therefore, finding a safe, effective and controllable collagen substitute has become an urgent problem to be solved.

[0008] Therefore, the present invention discloses a recombinant collagen protein on one hand, wherein the amino acid sequence of the recombinant collagen protein is shown as SEQ ID NO.1.

[0009] Preferably, the codon-optimized nucleotide sequence of the recombinant collagen of the present invention is as shown in SEQ ID NO.2.

[0010] In one aspect, the present invention further discloses an anti-aging composition, wherein the composition has a mass ratio formula of:

[0011] (1) Recombinant collagen: 5%;

[0012] (2) Sodium hyaluronate: 2%;

[0013] (3) Vitamin C: 0.5%;

[0014] (4) Coenzyme Q10: 0.3%;

[0015] (5) Vitamin E: 0.1%;

[0016] (6) Pure water: appropriate amount, adjust to 100%.

[0017] In one aspect, the present invention also discloses a method for preparing the composition, characterized in that the method comprises the following steps:

[0018] (1) Under clean conditions, dissolve the recombinant collagen in pre-sterilized PBS buffer and stir thoroughly until completely dissolved;

[0019] (2) gradually adding sodium hyaluronate, maintaining the temperature at about 20° C., and using a colloid mill to evenly disperse until a transparent gel solution is formed;

[0020] (3) Add vitamin C, coenzyme Q10 and vitamin E in order according to the ratio, and stir slowly to ensure that the ingredients are fully mixed and evenly distributed;

[0021] (4) Finally, the pH value was adjusted to 5.5-6.5 to obtain the final composition.

[0022] In one aspect, the present invention also discloses a use of the recombinant collagen in preparing an anti-aging composition.

[0023] In one aspect, the present invention also discloses a use of the anti-aging composition in preparing an anti-aging cream.

[0024] The present invention uses recombinant collagen and combines multiple anti-aging ingredients to develop an anti-aging composition with significant synergistic effects. Recombinant collagen has a structure highly similar to human collagen and excellent biocompatibility, and can effectively replace animal-derived collagen. Through modern genetic engineering technology, the recombinant collagen is expressed in a eukaryotic expression system and purified to ensure its high purity and high efficiency. Combined with sodium hyaluronate, vitamin C, coenzyme Q10 and vitamin E, which have been widely verified to have anti-aging effects, the composition of the present invention has significant advantages in anti-wrinkle, preventing collagen loss and anti-oxidation.

[0025] Most traditional anti-aging products use collagen extracted from animal tissues. Although they have certain moisturizing and elasticity-enhancing effects, they have problems such as unstable sources and high risk of allergic reactions. In contrast, the recombinant collagen used in the present invention is not only controllable in source and highly safe, but also has a specific molecular structure that is closer to human collagen and can effectively promote collagen synthesis in skin cells. The recombinant collagen expressed by genetic engineering technology has an optimized amino acid sequence, strong specificity and high purity, which effectively solves the limitations of traditional collagen.

[0026] The present invention is based on recombinant collagen and combines a variety of active ingredients with significant anti-aging effects, including sodium hyaluronate, vitamin C, coenzyme Q10 and vitamin E, to innovatively design a composite formula with synergistic effects. The mechanism of action of each ingredient complements each other, and together enhances the antioxidant, collagen synthesis and skin moisturizing abilities of the composition:

[0027] (1) Sodium hyaluronate: It absorbs a large amount of water, enhances the skin's moisturizing ability, forms a water-retaining barrier on the skin surface, improves skin hydration, and reduces the formation of wrinkles.

[0028] (2) Vitamin C: As a powerful antioxidant, it can neutralize free radicals, prevent skin cell aging caused by oxidative stress damage, and promote collagen synthesis.

[0029] (3) Coenzyme Q10: It is a fat-soluble antioxidant that can penetrate the skin barrier, reduce skin oxidative damage, maintain cell vitality, and participate in intracellular energy metabolism.

[0030] (4) Vitamin E: It has antioxidant and moisturizing properties, can protect cell membranes from free radical damage, and delay skin aging.

[0031] These ingredients work synergistically to not only increase skin hydration and elasticity, but also reduce the formation of wrinkles and damage caused by oxidative stress on the skin, demonstrating excellent anti-aging effects.

[0032] The present invention ensures uniform distribution of each component and maintains its biological activity unaffected through a reasonable preparation process:

[0033] (1) Recombinant collagen is expressed through advanced gene recombination technology. After purification, its three-dimensional helical structure is retained, ensuring its good compatibility with skin cells and biological activity.

[0034] (2) Low-temperature homogenization is used to avoid the destruction of bioactive ingredients by high temperature while maintaining the physical and chemical properties of hyaluronic acid and other active ingredients.

[0035] (3) The ingredients in the formula are precisely controlled in mass ratio, so that recombinant collagen, hyaluronic acid, vitamin C and other ingredients can complement each other and maximize their anti-aging effects.

[0036] Compared with the prior art, the recombinant collagen anti-aging composition of the present invention has the following significant advantages:

[0037] (1) High safety: The source of recombinant collagen is stable, avoiding the potential allergenicity of animal-derived collagen and having higher biocompatibility and safety.

[0038] (2) Significant anti-aging effect: Cell experiments have shown that the composition of the present invention can significantly increase the proliferation rate, antioxidant capacity and collagen synthesis of skin fibroblasts, showing excellent anti-aging properties.

[0039] (3) Innovative ingredient synergy: The present invention uses an optimized formula design and the synergistic effect of recombinant collagen with anti-aging ingredients such as hyaluronic acid and vitamin C to significantly improve the skin's moisturizing, elasticity and antioxidant capacity.

[0040] (4) The production process is simple and controllable: The preparation method of the present invention is simple and easy to operate, suitable for industrial production, and can effectively control the quality and biological activity of the product.

[0041] In summary, the present invention not only overcomes many limitations of existing anti-aging products, but also further expands the application of recombinant collagen in the anti-aging field through careful formula design and advanced preparation technology. It is widely used in the fields of medicine, cosmetics, etc., especially for the prevention and treatment of skin problems related to skin aging, wrinkle formation, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Western Blot detected the expression results of recombinant collagen, where 1 is recombinant collagen.

[0043] Figure 2 The results of SDS-PAGE detection of recombinant collagen, where 1 is recombinant collagen. DETAILED DESCRIPTION

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0045] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0046] Example 1: High-efficiency expression and purification of recombinant collagen

[0047] Short peptide (EEMQRR) is a botulinum toxin-like peptide that helps reduce the formation of expression lines by inhibiting nerve conduction and reducing muscle contraction. It is often used in anti-wrinkle products.

[0048] Therefore, when preparing COL1A1, we added the short peptide to its N-terminus. Therefore, the amino acid sequence of the recombinant collagen prepared by the present invention is shown in SEQ ID NO.1.

[0049] 1. Construction of expression vector: Amplify the nucleotide sequence of the recombinant collagen codon optimized by PCR (as shown in SEQ ID NO.2). Then, connect the amplified product with the pcDNA3.1(+) vector after digestion with EcoRI and HindIII, transform DH5α competent cells, and select ampicillin-resistant clones for plasmid extraction and sequencing verification.

[0050] 2. CHO cell culture: After CHO-K1 cells were revived, they were inoculated into a culture dish containing DMEM / F12 medium (10% FBS) and cultured at 37°C, 5% CO2 until the cell confluence rate reached 60-70%.

[0051] 3. Transfection: 1 μg plasmid DNA and 3 μL Lipofectamine TM 3000 reagents were mixed in Opti-MEM medium and allowed to stand for 15 minutes. The mixture was added to the culture dish containing CHO cells and incubated at 37°C for 6 hours, followed by replacement with fresh complete medium. After 48 hours, the medium containing 400 μg / mL of G418 was used for selection for 2-3 weeks until all non-transfected cells died.

[0052] 4. Screening and expansion of stable clones: Select surviving monoclonal CHO cells and expand them step by step until a cell line with stable expression of the human COL1A1 gene is obtained. After screening, a better CHO cell line was obtained, named CHO-COL1A1, which was expanded and cultured and stored in liquid nitrogen for future use.

[0053] 5. qPCR detection of COL1A1 mRNA expression: Extract total RNA from transfected CHO cells and synthesize cDNA using a reverse transcription kit. Use the following COL1A1-specific primers for qPCR detection. The primers are as follows:

[0054] Forward primer: 5′-GAGGGCCAAGACGAAGACAT-3′;

[0055] Reverse primer: 5′-GCCTCGTTGGAAACCTTCTT-3′.

[0056] qPCR conditions: initial denaturation: 95°C, 5 min; denaturation: 95°C, 10 sec; annealing: 60°C, 30 sec; extension: 72°C, 30 sec, 40 cycles.

[0057] The results showed that the CT value of COL1A1 was 19.4, and the CT value of GAPDH was 16.2, indicating that the expression of COL1A1 in CHO cells was significantly upregulated after transfection.

[0058] 6. Western Blot detection of COL1A1 protein expression: Total protein of CHO cells was extracted and separated by SDS-PAGE. The membrane was transferred to PVDF membrane and immunodetection was performed using anti-COL1A1 antibody (1:1000 dilution). ECL development was performed and a recombinant collagen band of about 160 kDa was observed ( Figure 1 ). This proves that COL1A1 protein is successfully expressed and the expression level is very high (the band is very bright).

[0059] 7. Recombinant collagen purification and identification: Use affinity chromatography columns to purify the expressed type I collagen and collect the purified protein samples. The molecular weight of the purified collagen was further confirmed by SDS-PAGE. The concentration of the purified collagen was quantitatively determined using the BCA method. The results showed that the concentration of the purified collagen was 3.8 mg / mL. By conversion, the expression level of the purified protein could reach 2 g / L. Therefore, the protein has a high expression yield, which provides a good foundation for industrial application. Electrophoresis results ( Figure 2 ) showed that the purified product was mainly a 160 kDa recombinant collagen band with a purity of about 95%.

[0060] 8. According to the above method, collagen without short peptide was also prepared, named collagen 1, and its purified expression level was about 0.5 g / L. This shows that the short peptide may also have the effect of increasing the expression of collagen.

[0061] Example 2: Preparation and application of anti-aging composition

[0062] 1. Preparation of anti-aging composition

[0063] 1.1 Ingredient ratio: According to the literature and preliminary experimental data, the ratio of each ingredient in the formula is optimized to ensure the synergistic effect of each component and enhance the anti-aging effect. The composition formula is as follows (all weight ratios):

[0064] (1) Recombinant collagen: 5%;

[0065] (2) Sodium hyaluronate: 2%;

[0066] (3) Vitamin C: 0.5%;

[0067] (4) Coenzyme Q10: 0.3%;

[0068] (5) Vitamin E: 0.1%;

[0069] (6) Pure water: appropriate amount, adjust to 100%.

[0070] 1.2 Preparation process

[0071] (1) Under clean conditions, dissolve the recombinant collagen in pre-sterilized PBS buffer and stir thoroughly until completely dissolved;

[0072] (2) gradually adding sodium hyaluronate, maintaining the temperature at about 20° C., and using a colloid mill to evenly disperse until a transparent gel solution is formed;

[0073] (3) Add vitamin C, coenzyme Q10 and vitamin E in order according to the ratio, and stir slowly to ensure that the ingredients are fully mixed and evenly distributed;

[0074] (4) Finally, the pH value was adjusted to 5.5-6.5 to obtain the final composition.

[0075] 1.3 At the same time, the collagen 1 prepared in Example 1 was used to prepare composition 1 as a control.

[0076] 2. In vitro anti-aging effect verification

[0077] 2.1 Cell proliferation assay (MTT assay)

[0078] In order to evaluate the promoting effect of the composition on the proliferation of human skin fibroblasts (HDFs), the MTT method was used for detection. HDFs cells were inoculated in a 96-well plate. After culturing for 24 hours, different concentrations of recombinant collagen composition (0.1%, 0.5%, 1.0%, 5.0%) were added, and a control group (containing only culture medium) was set up. Three replicates were set up for each group.

[0079] After 48 hours of culture, 10 μL of MTT solution was added to each well and incubated for 4 hours. Then 150 μL of DMSO was added to dissolve the formed crystals, and the absorbance value (OD value) was measured at a wavelength of 570 nm using a microplate reader to evaluate cell proliferation.

[0080] The experimental results show that as the concentration of the recombinant collagen composition increases, the proliferation rate of HDFs cells increases significantly. At a concentration of 1.0%, the cell proliferation rate increases by 30% compared with the control group, and at a concentration of 5.0%, the proliferation rate reaches 50%, and both are better than the effect of the composition 1 prepared by collagen 1 (see Table 2). This shows that the composition has a strong cell activity promoting effect.

[0081] Table 2 Cell proliferation assay results

[0082] Composition concentration 0.1％ 0.5％ 1.0％ 5.0％ Cell proliferation rate (%) 105％ 115％ 131％ 152％ Composition 1 concentration 0.1％ 0.5％ 1.0％ 5.0％ Cell proliferation rate (%) 95％ 101％ 111％ 126％

[0083] 2.2 Antioxidant capacity detection (SOD activity determination)

[0084] In order to detect the antioxidant capacity of the composition against oxidative stress, a SOD activity assay kit was used to analyze its protective effect on HDFs cells under oxidative stress. After HDFs cells were cultured in a 96-well plate for 24 hours, 0.5% and 5.0% concentrations of the composition were added, respectively, and cultured for 48 hours. Oxidative stress was then induced using H2O2, and intracellular SOD activity was detected after incubation for 30 minutes.

[0085] The experimental results show that the composition at 0.5% and 5.0% concentrations can significantly increase the SOD activity in HDFs cells. The SOD activity increased by 40% at a concentration of 5.0%, and was better than the effect of composition 1 prepared by collagen 1, indicating that the composition prepared by the present invention has significant antioxidant capacity (see Table 3).

[0086] Table 3 Antioxidant capacity test results

[0087] Composition concentration Control group 0.5％ 5.0％ SOD activity enhancement rate (%) 0 25％ 40％ Composition 1 concentration Control group 0.5％ 5.0％ SOD activity enhancement rate (%) 0 18％ 27％

[0088] 2.3 Detection of collagen gene expression promotion (RT-PCR)

[0089] In order to evaluate the effect of the composition on collagen gene expression, RT-PCR was used to detect the mRNA expression level of type I collagen (COL1A1) gene in HDFs cells. After 48 hours of cell culture, total RNA was extracted and reverse transcribed into cDNA, and real-time quantitative PCR was performed using collagen-specific primers to analyze the expression changes of COL1A1 gene after treatment with the composition.

[0090] RT-PCR results showed that the treatment with the composition at 1.0% and 5.0% concentrations significantly increased the expression level of the COL1A1 gene. The expression of COL1A1 mRNA at 1.0% concentration increased by 60% compared with the control group, and at 5.0% concentration increased by 110%, but the effect of the composition 1 prepared with collagen 1 was equivalent to or slightly better (see Table 4). This shows that the composition significantly promotes the synthesis of type I collagen and helps slow down the skin aging process.

[0091] Table 4 Detection results of collagen gene expression

[0092]

[0093] Example 3: Application of the composition

[0094] 1. Skin elasticity measurement: The anti-aging composition of the present invention was compared with a commercially available product. 20 volunteers used the cream product of the present invention twice a day for 4 consecutive weeks, and the results were tested using a skin elasticity meter, as shown in Table 5. The composition of the present invention has a better effect than the commercially available product (control group) (Johnson & Johnson, Hydro Boost Water Gel).

[0095] Table 5 Skin elasticity measurement experimental results

[0096] Product Type Elasticity value before use Elasticity value after 4 weeks of use Improvement rate Compositions of the present invention 58.2±4.3 72.5±3.9 24.6％ Commercially available products (control group) 59.0±3.8 65.2±4.1 10.5％

[0097] 2. Wrinkle improvement effect: The changes in facial wrinkles of volunteers were evaluated using a skin surface microscope, and the results are shown in Table 6. The composition of the present invention has a better effect than the commercially available product (control group) (Johnson & Johnson, Hydro Boost Water Gel).

[0098] Table 6 Wrinkle improvement effect test results

[0099] Product Type Wrinkle score before use Wrinkle score after 4 weeks of use Wrinkle reduction rate Compositions of the present invention 4.5±0.6 2.7±0.5 40％ Commercially available products (control group) 4.6±0.5 3.9±0.4 15％

[0100] The above results show that the recombinant collagen anti-aging composition of the present invention shows significant superiority in improving skin elasticity and reducing wrinkles, and has a higher anti-aging effect than commercially available products.

[0101] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A recombinant collagen, characterized in that: The amino acid sequence of the recombinant collagen is shown in SEQ ID NO.

1.

2. The recombinant collagen according to claim 1, characterized in that The nucleotide sequence of the recombinant collagen after codon optimization is shown in SEQ ID NO.

2.

3. An anti-aging composition, characterized in that: The mass ratio formula of the composition is: (1) The recombinant collagen described in claim 1: 5%; (2) Sodium hyaluronate: 2%; (3) Vitamin C: 0.5%; (4) Coenzyme Q10: 0.3%; (5) Vitamin E: 0.1%; (6) Pure water: appropriate amount, adjust to 100%.

4. A method for preparing the composition according to claim 3, characterized in that: The method comprises the following steps: (1) Under clean conditions, dissolve the recombinant collagen described in claim 1 in a pre-sterilized PBS buffer and stir thoroughly until completely dissolved; (2) gradually adding sodium hyaluronate, maintaining the temperature at about 20° C., and using a colloid mill to evenly disperse until a transparent gel solution is formed; (3) Add vitamin C, coenzyme Q10 and vitamin E in order according to the ratio, and stir slowly to ensure that the ingredients are fully mixed and evenly distributed; (4) Finally, the pH value was adjusted to 5.5-6.5 to obtain the final composition.

5. Use of the recombinant collagen as claimed in claim 1 in preparing an anti-aging composition.

6. Use of the anti-aging composition according to claim 3 in preparing an anti-aging cream.

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